Mutational analysis of flucytosine resistance in Candida glabrata

Abstract

The antifungal flucytosine (5-fluorocytosine [5FC]) is a prodrug metabolized to its toxic form, 5-fluorouracil (5FU), only by organisms expressing cytosine deaminase. One such organism is Candida glabrata, which has emerged as the second most common agent of bloodstream and mucosal candidiasis. This emergence has been attributed to the high rate at which C. glabrata develops resistance to azole antifungals. As an oral agent, 5FC represents an attractive alternative or complement to azoles; however, the frequency of 5FC resistance mutations and the mechanisms by which these mutations confer resistance have been explored only minimally. On RPMI 1640 medium containing 1 μg/ml 5FC (32-fold above the MIC, but less than 1/10 of typical serum levels), resistant mutants occurred at a relatively low frequency (2 × 10⁻⁷). Three of six mutants characterized were 5FU cross-resistant, suggesting a mutation downstream of the Fcy1 gene (cytosine deaminase), which was confirmed by sequence analysis of the Fur1 gene (uracil phosphoribosyl transferase). The remaining three mutants had Fcy1 mutations. To ascertain the effects of 5FC resistance mutations on enzyme function, mutants were isolated in ura3 strains. Three of seven mutants harbored Fcy1 mutations and failed to grow in uridine-free, cytosine-supplemented medium, consistent with inactive Fcy1. The remainder grew in this medium and had wild-type Fcy1; further analysis revealed these to be mutated in the Fcy2L homolog of S. cerevisiae Fcy2 (purine-cytosine transporter). Based on this analysis, we characterized three 5FC-resistant clinical isolates, and mutations were identified in Fur1 and Fcy1. These data provide a framework for understanding 5FC resistance in C. glabrata and potentially in other fungal pathogens.

FIG. 1.

Salvage pathway for cytosine (or 5FC) uptake and conversion to UMP (or 5-fluoro-UMP) in yeast (and in the case of 5FC, the downstream consequences on RNA, DNA, and protein synthesis). Also shown (in abbreviated form) are the alternative pathways for UMP production via the de novo pathway or uridine uptake.

FIG. 2.

(A) Alignment of Fcy1 sequences from C. glabrata (CgFcy1) and S. cerevisiae (ScFcy1). Residues involved in substrate binding (▵) or zinc binding and catalysis (▴), based on the ScFcy1 crystal structure (8, 13), are labeled. Residues that are mutated in 5FC-resistant C. glabrata (Table 2) are underlined, and the mutations are indicated (*, premature stop codon). Also shown are previously reported 5FC resistance-conferring mutations in Fcy1 orthologs of C. albicans (7), C. dubliniensis (18), C. lusitaniae (5), and C. glabrata (28), with the mutation preceded by Ca, Cd, Cl, and Cg, respectively. (B) Alignment of C. glabrata Fur1 (CgFur1) and T. gondii UPRT (TgUPRT) sequences (for space reasons, the 26 N-terminal residues of TgUPRT are not shown). Residues within the crystal structure (26) which bind the substrate phosphoribosyl pyrophosphate (▵) or the GTP regulator (⋄) and those which function as an active site general base (▴) or uracil hood (•) are labeled. Residues that are mutated in 5FC-resistant C. glabrata (Table 2) are underlined, and the mutations are indicated. Also shown are previously reported 5FC/5FU resistance-conferring Fur1 mutations in S. cerevisiae (11, 12), C. albicans (2, 7), and C. glabrata (1), with the mutation preceded by Sc, Ca, and Cg, respectively.